Project Summary A diverse array of cell types in mammalian skin together protects the organisms from insults, infection, and dehydration. Recent studies have begun to elucidate the interdependency of different cell types and the importance of cell-cell communication for the development and maintenance of a fully functional skin. Hair follicles, important appendages of the epidermis, cycle between a growth phase (anagen) and a rest phase (telogen). Hair follicle cells are not the only cell type that is different between an anagen and a telogen skin: upon anagen entry, the dermis increases significantly through proliferation of dermal fibroblasts embryonically and expansion of the dermal adipose layer postnatally, resulting in skin that is thicker and more resilient than telogen skin. Anagen skin also exhibits faster wound-healing than telogen skin. Despite these known differences, however, there remains a fundamental gap in understanding of the signaling pathways and specific cell-cell communications that lead to these changes. This gap poses a significant impediment to effective therapeutic intervention in a variety of areas including wound repair, congenital dermal disorders, and atrophic skin following chemotherapy or skin grafting. Transit-amplifying cells (TACs) are generated by long- term stem cells as an intermediate population that produce downstream progeny. Hair follicle TACs (HF-TACs) are an anagen-specific population. Our preliminary data indicate that HF-TACs play a key role in these anagen-specific skin changes through a mechanism involving Sonic Hedgehog (SHH). The overall objective of this proposal is to elucidate the cellular mechanisms by which HF-TAC-specific SHH mediates anagen-specific skin changes and to identify the signal-receiving cells that are responsible for dermal thickening and faster wound healing in anagen skin. Our central hypothesis is that the SHH signaling, operating through the key downstream mediator, Smoothened, orchestrates the changes that make anagen skin thicker, more resilient, and faster healing. To test this hypothesis rigorously, a systematic in vivo approach will be taken to examine the direct requirement of SHH signaling in different cell types in the skin by knocking-out essential downstream mediators of SHH and comparing the phenotypes of these mutants to phenotypes of Shh knockout skin. The cell types to be examined include hair follicles, dermal fibroblasts, mature adipocytes, and adipocyte precursors. We will use cell-type specific inducible or constitutive Cre lines that we have verified extensively for expression patterns and effectiveness. We expect that the experiments outlined in this proposal will elucidate a physiologically relevant pathway by which anagen skin becomes thicker and heals better. We anticipate that the knowledge gained will identify critical cell types and/or signals that might be harnessed therapeutically in pathological conditions. The results may also identify novel regulatory functions exerted by TACs on the surrounding microenvironment, which would likely impact studies of TACs in other systems.